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研究生:王鈞民
研究生(外文):Chun-Min Wang
論文名稱:阿拉伯芥中的NHP25基因之選殖、鑑定及其於生長發育過程之功能分析
論文名稱(外文):Isolation, identification and functional characterization of NHP25 in Arabidopsis development and growth
指導教授:王國祥
指導教授(外文):Co-Shing Wang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:58
中文關鍵詞:切割刺激因子生長發育過程切割因子
外文關鍵詞:CstFCPSFCF
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真核生物基因表達的過程包含所轉錄之mRNA、後轉錄、轉譯和後轉譯修飾。在mRNA修飾過程中,polyadenylation是在3’端上加poly A,但其分子機制在植物中仍然不清楚。在動物與酵母菌的研究指出,mRNA在加上polyA之前,需經由切割刺激因子複合物 (Cleavage stimulation factor complex, CstF complex)辨認mRNA上的特定序列,然後吸引其它蛋白共同執行mRNA切割作用。首先我們實驗室主要研究課題是藉由模式植物阿拉伯芥探究花粉發育和授粉相關基因之選殖及功能分析。利用生物資訊學的方式進行大規模基因選殖,再經由一系列篩選後,我們得到幾個可能與雄配子發育有關的基因,並利用其相對之T-DNA插入突變株進行後續功能分析。其中一個T-DNA 插入突變株無法產生homozygote的後代,我們命名為Non Homozygotic Progeny 25 (NHP25),進一步比對flanking 序列發現此基因為切割刺激因子複合物的一個次單元,AtCstF-64。在所分析該基因數個T-DNA插入突變株,共出現兩個各別具性狀且不孕的同型合子T-DNA插入突變株(nhp25-1及nhp25-5),例如:晚開花、生長緩慢、鋸齒葉、畸形花等。利用互補實驗證實NHP25能完全回復nhp25-1突變株的生長缺陷。我們利用基因晶片找到許多受到NHP25調控的基因,其中發現了兩個與開花調控有關的基因(SPL3與SPL4)會在突變株中表現量降低。結果顯示NHP25可能藉由這兩個基因來參與開花時間的調控。

Eukaryotic gene expression is a complex process includes transcriptional, post-transcriptional (e.g. pre-mRNA processing), translational, and post-translational regulations (e.g. protein modifications). During pre-mRNA processing event, polyadenylation is mediated by adding polyadenosine into newly formed 3’ end of mRNA after nascent mRNA cleavage, but its molecular mechanism is still unclear in plant. However, recent studies indicated that the nascent mRNA cleavage is mediated by a novel cleavage stimulation factor (CstF) complex that does not directly cleavage RNA but assists other proteins to process RNA through recognizing specific cis-element in nascent mRNA itself. Our long-termed interest is to identify and functionally investigate the genes/proteins involved in pollen development and pollination. By taking advantage of tremendous resources generated from the studies of Arabidopsis, we used bioinformatics and data-mining approach at the genome level to search the T-DNA insertion mutants with defective male gametophyte development. One of mutants that could not produce homozygotic progeny is caused by T-DNA insertional disruption of Arabidopsis gene Non Homozygotic Progeny 25 (NHP25), which was confirmed by flanking sequencing. Interestingly, among all the characterized Arabidopsis T-DNA line of NHP25, two of them (nhp25-1 and nhp25-5) might be leakage and showed pleiotropic phenotypes in sterile homozygote mutants including late flowering, draft growth, jigsaw rosette leave, abnormal flower morphology, and seed abortion. Complementary assay shows fully complement in two different T-DNA insertion alleles. The microarray experiment surveyed the genes which are affected in nhp25 mutant and found two flowing time regulated genes, SPL3 and SPL4 are significantly downregulated in nhp25 mutant, suggesting the NHP25 might regulate flowering time through controlling SPL3 and SPL4 expression.

緒言………………………………….........................1
前人研究…………………………...........................2
材料與方法……………………………………................11
結果……………………………………......................19
一. 基因型鑑定及同型合子植株性狀的觀察…………….19
二. 各物種中CstF-64的序列比對和演化樹………………20
三. nhp25-1互補實驗分析…………………………………21
四. NHP25在各器官間表現量分析…………………………21
五. NHP25座落位置 ( localization ) 分析……………22
六. 野生型植株及nhp25-1同型合子植株之生物微晶片分析結果…..................................................23
討論…………………………………………………………………24
一. nhp25-1~5基因型鑑定和性狀之探討…………………24
二. 各物種中CstF-64的序列比對探討……………………25
三. 35S和NHP25啟動子互補株之探討…………………….25
四. NHP25座落位置之探討…………………………………26
五. NHP25-1微矩陣分析之探討……………………………26
六. 哺乳動物、酵母菌及阿拉伯芥3’修飾的比較………27
圖表…………………………………………………………………29
一. 各T-DNA突變株的分離率………………………………29
二. NHP25-1影響基因表現量下降的基因…………………30
三. NHP25-1影響基因表現量上升的基因..............34
四. 引子列表……………………………………………...37
五. NHP突變株的篩選………………………………………40
六. NHP25基因和T-DNA插入位和 nhp25-1和nhp25-5的性狀…………............................................41
七. nhp25-1和nhp25-5突變株的NHP25基因的表達圖……42
八. 真核生物NHP25基因之比對……………………………43
九. 真核生物NHP25基因之演化樹…………………………44
十. nhp25-1轉殖植株性狀…………………………………45
十一. NHP25在各器官的表達………………………………..46
十二. 利用原生質體作NHP25座落位子的分析………………47
十三. 利用原生質體作CstF-50座落位子的分析……………48
附圖…………………………………………………………………49
參考文獻……………………………………………………………53

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